Sensing apparatus having rotating optical assembly

ABSTRACT

An apparatus for optical sensing of samples includes an optical source, an optical assembly being rotatable about an axis, a sample holder, and a detector. The optical assembly rotates, allowing the sensing apparatus to sense results from plural locations on a sample without moving the sample. Moving the sample in a linear direction while rotating the optical assembly allows sensing of an entire sample containing multiple test sites, such as a biochip. An optical assembly containing mirrors to direct light from the optical source to the sample is provided. Preferably, light enters the optical assembly along the axis of rotation. Sensing methods consistent with the invention are also described.

BACKGROUND

1. Field

The present invention relates to the field of sample sensing.

2. Description of Related Art

Conventional sensing apparatuses use one or more optical sources to readsamples. Biochip scanners, for example, use laser light to irradiate achemical or biological sample, which, depending on material in thesample, responds by fluorescing. By detecting the light emitted by thesample, conventional apparatuses can identify specific materials in thesample, as well as the amount of those materials present. In oneapplication, sensing apparatuses may be used, for example, to captureresults from multiple reactions in real time by rapidly scanning thesample surface.

The samples may include, for example, a biochip. As appreciated by thoseof skill in the art, a biochip is a collection of test sites arranged ona solid substrate. These biochips allow scientists to monitor multiplechemical or biochemical reactions simultaneously, and have applicationsin genetics, toxicology, biochemical, protein, and other research areasrelated to chemistry and biochemistry. A biochip may contain thousandsof individual test sites, each representing the outcome of an individualreaction.

Typically, conventional sensing apparatuses irradiate and monitor onlyone test site at time until each test site on the sample has beenexposed. Conventional biochip scanners, for example, move either thesample or the body of the sensing apparatus in a linear or grid fashion,directing light to each individual test site on the sample. In thismanner, the sensing apparatus scans each individual test site todetermine whether it contains the material or materials to be sensed.

Sensing and mapping materials on samples with multiple test sites inreal-time requires sensing apparatuses capable of quickly directing andreceiving light from distinct locations on the sample. Quicklycollecting data from samples containing thousands of individualexperimentation sites, like biochips, speeds research and saveslaboratory resources. To effectively capture experimental results,therefore, it is desirable to provide sensing apparatuses and methodsthat hasten sample sensing.

Moreover, sensing apparatuses and methods capable of detecting materialson samples containing multiple test sites arranged in a non-rectilinearpattern create flexibility for users. Samples containing multiple testsites arranged in spirals, circles, or arcs, for example, provideenhanced sensing opportunities. While arranging test sites in a patternother than a grid allows for a higher density of test sites on thesample, conventional sensing apparatuses are not configured to readsamples laid out in this manner.

Additionally, it is desirable to create apparatuses and sensing methodsthat sense samples without moving the samples in rectilinear fashion, asdone in conventional sensing apparatuses. Such apparatuses and methodscan scan samples containing non-rectangular test sites.

BRIEF SUMMARY

A sensing apparatus is provided, which comprises a sample holderconfigured to receive a sample, an optical source, an optical assemblybeing rotatable about an axis, and a detector. The optical assembly isspaced from the sample holder, and is configured to receive excitationlight outputted by the optical source and to direct the excitation lightto a first location on the sample when the optical assembly is in afirst position. The optical assembly also directs the excitation lightto a second location on the sample when the optical assembly is rotatedto a second position. The detector is configured to receive emittedlight from the first and second locations on the sample, which isfluoresced in response to the first light.

A sensing method is also provided, which comprises generating excitationlight, rotating an optical assembly to a first position, directing theexcitation light through an optical assembly to a first location on asample, detecting first emitted light, rotating the optical assembly toa second position, directing the excitation light through the opticalassembly to a second location on the sample, and detecting secondemitted light.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments consistentwith the invention and together with the description, serve to explainthe principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a first sensing apparatus consistentwith an embodiment of the invention;

FIG. 2 is a schematic diagram of a second sensing apparatus consistentwith an additional embodiment of the invention;

FIG. 3 is a schematic diagram of a third sensing apparatus consistentwith a further embodiment of the invention;

FIGS. 4A and 4B are schematic diagrams of an optical assembly consistentwith an embodiment of the invention.

FIG. 5 is a flowchart of a sensing method consistent with anotherembodiment of the invention;

FIG. 6 is a flowchart of a sensing method consistent with an additionalembodiment of the invention.

FIGS. 7A and 7B illustrate light-interruption plates for use in sensingapparatuses consistent with embodiments of the invention;

FIGS. 8A and 8B are schematic diagrams of the sensing apparatusillustrated in FIG. 2.

FIGS. 9A and 9B are schematic diagrams of a variation of the sensingapparatus illustrated in FIG. 2, consistent with an embodiment of theinvention.

FIG. 10 is a schematic diagram of a rotational sensing circle consistentwith sensing apparatuses consistent with embodiments of the invention.

FIGS. 11, 12, 13, 14, and 15 are schematic diagrams showing operationalmodes for sensing apparatuses consistent with embodiments of theinvention.

FIG. 16 is a schematic diagram of a control circuit for use in sensingapparatuses consistent with embodiments of the invention.

FIG. 17A-17C are schematic diagrams illustrating methods of creatingimages representing the light emitted by a sample, consistent withembodiments of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference will now be made in detail to exemplary embodiments consistentwith the invention, illustrated in the accompanying drawings. Whereverpossible, the same reference numbers will be used throughout thedrawings to refer to the same or like parts.

FIG. 1 illustrates a sensing apparatus 600 consistent with the presentinvention. Sensing apparatus 600 comprises an optical source 10, anoptical assembly 30, a detector 80, and a sample holder 100. A sample90, including a biochip, for example, is loaded into sample holder 100.As further shown in FIG. 1, sensing apparatus 600 further includes anoptical filter 20, an emission filter 60, and a focusing lens 70.Components of sensing apparatus 600 will next be described in greaterdetail.

Optical source 10 outputs excitation light 12 to optical filter 20.Optical source 10 preferably comprises a laser, in which case excitationlight 12 constitutes a laser beam having a single wavelength and phase.The preferable wavelength of excitation light 12 depends on sample 90.For example, green laser light is used to sense materials including Cy3dye, while red laser light is used to sense materials including Cy5 dye.Both of these dyes are known and can be provided in conventionalbiochips. Lasers suitable for use in a sensing apparatus include, forexample, the model LVI-VA-532-30 laser, commercially available from LVITechnology Co., Ltd. (typically used with Cy3 dye) or the LM-6535GRlaser, commercially available from Lanics Co., Ltd (typically used withCy5 dye).

Optical assembly 30, which is spaced from sample holder 100, receivesexcitation light 12 reflected from filter 20 and directs excitationlight 12 to sample 90. Optical assembly 30 is rotatable about axis 45,so that excitation light 12 can be directed to multiple locations onsample 90 without moving sample 90. Optical assembly 30 can beconfigured to rotate in a full circle, a semicircle, or an arc.

Optical assembly 30 preferably includes one or more mirrors to directexcitation light 12 to sample 90. In FIG. 1, for example, opticalassembly 30 comprises a first mirror 31 and a second mirror 32. Firstmirror 31 is located on axis 45, the axis about which optical assembly30 rotates. Second mirror 32 is located a distance apart from mirror 31along axis 46. Optical module 30 receives excitation light 12 along axis45, mirror 31 then reflects excitation light 12 toward mirror 32, whichin turn, directs excitation light 12 to sample 90 along axis 46.

Preferably, mirror 31 is arranged at substantially 45° relative to axis45. In this preferred arrangement, first mirror 31 reflects excitationlight 12 in a direction substantially perpendicular to axis 45. Secondmirror 32, located a distance away from first mirror 32, reflectsexcitation light 12 so that it travels in a direction substantiallyparallel to axis 45. Mirror 32 is arranged at substantially 45° relativeto axis 46 to reflect excitation light 12 to objective lens assembly 40,as discussed in greater detail below. As further shown in FIG. 1, axes45, 46 are substantially perpendicular to the surface of sample 90. Ifdesired, however, axes 45, 46 may be oriented at different anglesrelative to the surface of sample 90, as well as each other.

Moreover, excitation light 12 need not enter optical assembly 30 alongaxis 45. Configurations in which light optical assembly 30 receivesexcitation light 12 along an axis other than axis 45 are consistent withthe present invention. For example, mounting optical source 10 tooptical assembly 30 would allow light to enter the optical module alongan axis other than axis 45, such as, for example, axis 46. Such aconfiguration could eliminate the need for mirrors 31, 32.

Preferably, mirrors 31, 32 are of a type well known in the art, such as02MFG00 mirrors, which are commercially available from Melles Griot,Inc.

Due to the rotation of optical module 30, second mirror 32 follows anarc about axis 45. The distance between mirrors 31 and 32 defines theradius of this arc. This arc may constitute a semi-circle or a fullcircle, and can be any appropriate length.

Optical assembly 30 may further comprise an objective lens assembly 40,which includes one or more lenses. Objective lens assembly 40 may focusexcitation light 12, limiting the beam of light transmitted to sample 90to a desired size, so that a desired portion of sample 90 may beexposed. Also, once focused, excitation light 12 has a greater intensitythan the input to assembly 30. The resulting fluorescence of sample 90may thus also increase, thereby facilitating more accurate measurements.

Objective lens assembly 40 may also collimate emitted light 50, emittedwhen sample 90 fluoresces, into a beam of light. Persons of skill in theart will recognize how to design and construct objective lens assembliesexhibiting desirable characteristics.

Sensing apparatus 600 further comprises detector 80, which detectsemitted light 50. Detector 80 converts received light into an electricalsignal, which a user or computer can store and/or analyze. Detector 80may comprise, for example, a photo multiplying tube or an image sensor,such as a charge-coupled device (CCD). An exemplary photo multiplyingtube consistent with this application is the H6779 tube, commerciallyavailable from Hamamtsu Photonics, K.K. Suitable charge-coupled devices,such as model number KAF0261E manufactured by Eastman Kodak Corporation,are well known in the art.

Sample holder 100 holds sample 90 in place for sensing. As discussedabove, the rotation of optical module 30 allows sensing apparatus 600 todirect excitation light 12 to multiple locations on sample 90 withoutmoving sample 90. Thus, sample holder 100 may remain in one location.

Preferably, however, sample holder 100 moves along a substantiallystraight line (hereinafter “in a linear direction”). Moving sample 90 ina linear direction, when combined with the rotation of optical module30, allows sensing apparatus 600 to direct excitation light 12 to alllocations on sample 90 and to receive emitted light 50 from alllocations on sample 90. In this manner, sensing apparatus 600 can createa two-dimensional image of the sample from the electrical signalsgathered by detector 80. Thus, the sensing apparatus 600 can quicklyscan samples, such as biochips having the “2-dimension & large” format.In addition, by moving sample 90 and optical assembly 30 in both linearand rotational directions relative to one another, sensing apparatus 600decreases sensing time and enables continuous sample scanning. Personsof skill in the art will perceive other advantages of thisconfiguration.

Linear stage module 110 moves sample holder 100 in a linear direction.Preferably, linear stage module 110 comprises a stepper motor configuredto move sample 100 in a linear direction substantially perpendicular toaxis 45 in a stepwise fashion. Linear stage module may include, forexample, a stepper motor such as the model KH42JM2-901 stepper motor,commercially available from Japan Servo Co., Ltd. Moreover, sampleholder 100 may remain stationary while the rest of sensing apparatus 600moves in a linear direction in addition to the rotating motion discussedabove.

Optionally, movement of sample holder 100 may comprise a non-linearmotion and/or continuous motion. Non-linear motion may be desirablewhere sample 90 comprises non-rectangular test sites or test sitesarranged in a manner other than a rectilinear grid. Where sample holder100 moves in a non-linear manner, Sensing apparatus 600 would includeanother apparatus, such as a four-bar linkage, to move sample holder 100instead of linear stage module 110.

Preferably, sample 90 includes a biochip. Sensing apparatus 600 candirect excitation light 12 and detect emitted light 50 from multiplelocations on sample 90, making sensing apparatus 600 well-suited for usewith biochips or other samples having multiple test sites.

Sensing apparatus 600 further comprises an optical filter 20, configuredto reflect excitation light 12. When excitation light 12, generated bylight module 10, reaches optical filter 20, optical filter 20 reflectsexcitation light 12 toward optical module 30. In particular, whenexcitation light 12 includes of a particular wavelength or band ofwavelengths, optical filter 20 can be selected to reflect substantiallyall of excitation light 12 toward optical module 30. This provesparticularly useful where excitation light 12 constitutes a laser beam.

In at least some configurations, optical filter 20 does not reflectemitted light 50, emitted by sample 90. Indeed, in exemplary sensingapparatus 600, emitted light 50 passes through optical filter 20 beforereaching detector 80. Excitation light 12 and emitted light 50 are ofdistinct wavelengths, allowing sensing apparatus 600 to separate themusing optical filter 20. Preferably, optical filter 20 is a known filterhaving a coating configured to reflect light at the wavelength ofexcitation light 12, but to permit other wavelengths to pass.

Sensing apparatus 600 further comprises optical filter 60, used toattenuate light of unwanted wavelengths from emitted light 50. Opticalfilter 60 allows only light having a desired wavelength to pass through.Thus, optical filer 60 substantially attenuates any excitation light 12,generated by optical source 10, reflected off of sample 90 and mixed inwith emitted light 50 after passing through optical filter 20. Opticalfilter 60 thus may ease signal processing by substantially attenuatingany extraneous excitation light 12. The 51007m optical filter,commercially available from Chroma Technology Corporation, is an exampleof an optical filter consistent with optical filter 60.

Focusing lens 70 is configured to enhance the signal-gatheringperformance of detector 80. Since detector 80 measures the relativeintensity of light 50, detector 80 does need not be positioned at thefocal length of focusing lens 70. Placing a pinhole 71 at the focalpoint of focusing lens 70 allows for enhanced signal-to-noise ratio.Additional types and configurations of focusing and signal-processingapparatuses will occur to those of skill in the art.

The operation of sensing apparatus 600 will now be briefly describedwith further reference to FIG. 1.

Excitation light 12, output from optical source 10, is reflected byoptical filter 20 toward optical assembly 30.

Next, excitation light 12 enters optical assembly 30, traveling alongaxis 45. Mirror 31 reflects excitation light 12, directing it towardmirror 32. Mirror 32 reflects excitation light 12, toward objective lensassembly 40 along axis 46. Objective lens assembly 40, in turn, focusesexcitation light 12, directing only a desired portion of excitationlight 12 to sample 90.

Excitation light 12 excites sample 90, causing it to fluoresce and emitlight 50. The amount of emitted light 50 emitted by sample 90, if any,may depend on the result of an experiment performed on sample 90. Due toa biological or biochemical reaction, for example, sample 90 may containa particular material, which fluoresces in response to excitation light12.

Optical assembly 30 receives emitted light 50 through objective lensmodule 40, which collimates and directs emitted light 50. Mirror 32reflects emitted light 50, directing it toward mirror 31, which, inturn, reflects emitted light 50 along axis 45 to filter 20.

After exiting optical assembly 30, emitted light 50 passes throughoptical filter 20, which reflects any excitation light 12 that reflectedoff of sample 90 and was collimated along with emitted light 50 byobjective lens assembly 40.

Emitted light 50 next passes through optical filter 60, which attenuatesresidual excitation light 12 that passes through optical filter 20.After passing through optical filter 60, emitted light 50 reachesfocusing lens 70 and pin hole 71, which, as described above, enhance thesignal-to-noise ratio of emitted light 50 to maximize detection bydetector 80.

Finally, detector 80 detects emitted light 50. Detector 80 converts thedetected light into an electrical signal, which a user or computer canstore and/or analyze.

As discussed above, optical assembly 30 is rotatable about axis 45. Asillustrated in FIG. 4A, at a first position, optical assembly 30 directsexcitation light 12 to a first location 91 on sample 90 and receivesemitted light 50 from first location 91 on sample 90. When opticalassembly 30 rotates to a second position, illustrated in FIG. 4B, itdirects excitation light 12 to a second location 92 on sample 90 andreceives emitted light 50 from second location 92 on sample 90. Thus,over time, as optical assembly 30 rotates, excitation light 12 isdirected to plural locations on sample 90. Moreover, sample holder 100moves in linear direction 95 relative to axis 45, allowing sensingapparatus 600 to direct excitation light 12 to all locations on sample90.

As illustrated in FIG. 4A and FIG. 4B, first location 91 and secondlocation 92 may be located substantially opposite one another on acircle defined by the rotation of optical assembly 30 about axis 45.When first location 91 and second location 92 are located in thismanner, sensing apparatus 600 senses emitted light 50, 61 from each oflocations 91, 92 during a single rotation of optical assembly 30. Bysensing from plural locations on each rotation of optical assembly 30,the time required to sense emitted light from all desired locations onsample 90 decreases. While this configuration of sensing apparatus 600is preferable, first location 91 and second location 92 may be locatedadjacent to one another on sample 90, or in any other desiredconfiguration. Moreover, it may be possible to sense emitted light frommore than two locations on sample 90 with each rotation of opticalassembly 30. To differentiate emitted light 50 from emitted light 61, itmay be necessary to adjust the shutter time of detector 80.

As illustrated in FIG. 17A, optical assembly 30 preferably rotates aboutaxis 45, in a direction indicated by arrow 301, with a substantiallyconstant angular speed. This constant angular speed allows sensingapparatus 600 to identify the plural locations on sample 90 from whichit senses emitted light. The rotation of optical assembly 30 about axis45 defines rotational sensing circle 200, with a radius v extending fromaxis 45 to axis 46.

Preferably, sensing apparatus 600 detects emitted light from multipleregions on sample 90, each region typically having a square shape withsides substantially equal to the distance that sample 90 travels duringeach a full rotation of optical assembly 30 around axis 45. Region 225,illustrated in FIG. 17A, is an example of such a region and ispositioned where rotational sensing circle 200 meets reference line 220.Reference line 220 is a reference line, drawn with dashed lines,substantially parallel to direction 95 and passing through the middle ofsample 90. As shown in FIG. 17A, reference line 221, a second referenceline for purposes of this discussion, is substantially orthogonal toreference line 220 and also passes through the middle of sample 90.Reference lines 220, 221 are used for purposes of discussion, but maynot necessarily be present on sample 90.

Preferably a computer, not shown, is coupled to detector 80 and capturesmultiple pixels, each of which typically represents light emitted by aparticular region on sample 90. To “capture” each pixel, the computerreads an output from detector 80 and stores information—thepixel—representing the light detected by detector 80 for later use. Thecomputer later uses these multiple captured pixels to create an image ofthe light emitted by sample 90. To create an accurate image, thecomputer determines the region on sample 90, such as region 225, towhich each captured pixel corresponds and places the pixel in its properplace in the image. Preferably, the image has a uniform resolution inrectilinear coordinates.

The computer may use a timer and the constant angular speed at whichoptical assembly 30 rotates to correlate pixels captured from detector80 with regions on sample 90 to create the desired image of lightemitted by sample 90. The computer can create the desired image inmultiple ways, such as varying the timing it uses to capture pixels fromdetector 80.

For example, in a first preferable method of creating an image of lightemitted by sample 90, the computer captures pixels corresponding toregions located at equal angular increments around rotational sensingcircle 200. Because the angular speed of rotation of optical assembly 30is constant, the computer waits a predetermined period of time betweenthe capture of each individual pixel, and thus, the computer capturespixels at a constant frequency.

The region on sample 90 corresponding to each pixel is characterized by(1) an angle a measured between reference line 220 and radius v at thetime the computer captures the pixel and (2) the position of sample 90as it moves in direction 95. Angle a and the length of radius v, definepolar coordinates for each individual region in relation to the centerof rotational sensing circle 200. These polar coordinates allow for easytranslation to an orthogonal coordinate system, if desired.

Capturing pixels from regions at equal angular increments aroundrotational scanning circle 200 decreases the rectilinear space betweenregions on sample 90 as angle a increases to 90 degrees. Thus, regionscorresponding to captured pixels near reference line 220 are spaced lesstightly than regions located further from reference line 220. To ensurean adequate image with uniform resolution throughout, the computer maycapture additional pixels, by increasing the frequency of pixel capture.These additional pixels may include pixels representing overlappingregions on sample 90, particularly for regions located near the edges ofsample 90. The computer will then average these “oversampled” pixelstogether to create an image with a desired resolution. Pixelscorresponding to regions near the center of sample 90 will not requireaveraging, while pixels captured as angle a increases will beoversampled and, thus, be averaged by the computer. Such oversamplingand averaging procedure can counteract poor image quality that wouldotherwise result from the differences in distances between pixels.Accordingly, an image having a uniform resolution in a rectilinearcoordinate system can be obtained. Using this oversampling correction,however, typically captures and averages a high number of samples takenas the angle between radius v and reference line 220 increases toward 90degrees.

A second preferable method of creating an image of light emitted bysample 90 comprises detecting plural pixels corresponding to regionspositioned at intersections of the rotational sensing circle 200 and aseries of equidistant lines substantially parallel to reference line220. FIG. 17B illustrates an example of a series of such equidistantlines 220 and 230-235. To sample light emitted by regions 225 and240-245, detector 80 decreases the time it waits between capturingpixels as optical assembly 30 approaches the edges of sample 90. Forexample, detector 80 waits less time between detecting pixelscorresponding to regions 244 and 245 than it does between pixelscorresponding to regions 243 and 225. Using this method, sensingapparatus captures a set of pixels corresponding to regions distributedequidistantly in a direction parallel to reference line 221, simplifyingtranslation to an orthogonal grid. Conversely, in a directionparalleling reference line 220, the regions have no uniform spacing andare further apart the further optical assembly 30 is from reference line220. To create an image having uniform resolution, the computerinterpolates the pixels it captures. To interpolate between a first andsecond pixel, the computer can, for example, read a portion of the firstpixel closest to the second pixel, read a portion of the second pixelclosest to the first pixel, and use the portions of the first and secondpixels to approximate what the space between the first and second pixelsshould look like. The computer may also simply divide the first pixel,using a portion of it to approximate the image required for an adjacentpixel. Thus, as optical assembly 30 rotates closer to the edges ofsample 90, the computer uses the captured pixels to create the desiredimage having a uniform resolution.

A third preferable method of creating an image of light emitted bysample 90, illustrated in FIG. 17C, also detects a plurality of pixelscorresponding to regions positioned at the intersections of therotational sensing circle 200 and a series of equidistant, substantiallyparallel lines 250-253, which parallel reference line 221, not referenceline 220. This method captures pixels corresponding to regions 225 and255-259, which are distributed equidistantly in the in a directionparalleling reference line 220, but not in a direction parallelingreference line 221. Again, the computer interpolates between detectedpixels when creating the image to create uniform resolution. Usingeither the second or third methods described herein requirescomparatively less oversampling than the first method. FIGS. 17B and 17Cillustrate only exemplary spacing of equidistant lines, and the sensingrequirements of particular applications will dictate the requireddetailed of an image of the light emitted by sample 90, if any.Moreover, other imaging methods and pixels having a shape other thansquares are contemplated herein.

FIG. 2 illustrates sensing apparatus 601, also consistent with thepresent invention. Like sensing apparatus 600 described above, sensingapparatus 601 comprises an optical source 10, an optical filter 20, anoptical assembly 30, an emission filter 60, a focusing lens 70, and adetector module 80. In addition to these components, sensing apparatus601 further comprises an optical source 11, an optical filter 22, and anoptical chopper 120, which are described below.

Preferably, optical source 11 includes a laser, which outputs excitationlight 13 having a wavelength different than that of excitation light 12.In this configuration, excitation light 13 causes sample 90 to fluoresceand emit light 61, which has a wavelength different from the wavelengthof the emitted light 50 emitted by sample 90 in response to excitationlight 12. Thus, sample 90 emits light 50 at a first wavelength and emitslight 61 at a second wavelength in response to exposure to excitationlight 12 and excitation light 13, respectively.

As with excitation light 12, the optimal wavelength of excitation light13 depends on sample 90. Persons of skill in the art typically use laserbeams with different wavelengths to sense experimental results fromsamples including Cy3 dye and Cy5 dye, respectively. Because sensingapparatus 601 contains two optical sources 10, 11, it can sense samplesdyed with two different dyes. Lasers suitable for use in optical source11 are generally of the same type and grade as those suitable for use inoptical source 10. Below, optical source 10 preferably includes a greenlaser, such as the LVY-VA-532-30 laser, and optical source 11 preferablyincludes a red laser, such as the LM-6535GR laser.

Alternatively, Optical source 11 and optical source 12 can includelasers producing light having the same wavelength. In this alternativeconfiguration, for example, optical source 11 and optical source 12could each include a green laser. In this configuration, sensingapparatus 601 would be used to sense samples dyed with a single dye. Aswill be explained below, using two optical sources, as in sensingapparatus 601, as opposed to a single optical source, as in apparatus600, allows the sensing apparatus to sense all desired results on agiven sample more quickly.

Optical filter 22, like optical filter 20, is configured to reflectlight having a particular wavelength, but allows light having otherwavelengths to pass through. In particular, optical filter 22 preferablyhas a coating, which reflects excitation light 13, but allows emittedlight 50, 61 to pass through. Preferably, Optical filter 20 is theQ555lp optical filter, commercially available from Chroma TechnologyCorporation. Optical filter 22 is preferably the 61005bs optical filter,also available from Chroma Technology Corporation.

As further shown in FIGS. 2 and 8, sensing apparatus 601 includes anoptical chopper 120, which comprises a light-interruption plate mountedsubstantially perpendicular to a rotating shaft. Optical chopper 120 isconfigured to allow one of excitation light 12 or excitation light 13 toreach sample 90 at a time. As it rotates, optical chopper 120 moves froma position that interrupts excitation light 12 to a position thatinterrupts excitation light 13 and back again. In this manner, each ofexcitation light 12 and excitation light 13 are directed to sample 90for a portion of time, such that excitation light 12 and excitationlight 13 do not simultaneously expose the same location on sample 90.Preferably, optical chopper 120 rotates at the same rate as opticalassembly 30.

As mentioned above, optical chopper 120 prevents excitation light 12 andexcitation light 13 from simultaneously exposing the same location onsample 90. Thus, optical chopper 120 enables detector 80 to distinguishemitted light 50, emitted in response to excitation light 12, fromemitted light 61, emitted in response to excitation light 13. Opticalchopper 120 may also be provided in a sensing apparatus having multipledetectors, as discussed below and illustrated in FIG. 3.

FIG. 7 illustrates two exemplary light-interruption plates for use asoptical chopper 120 consistent with the present invention.Light-interruption plate 122, illustrated in FIG. 7A, is fixed to, androtates with, shaft 121. An open region 123 allows one of excitationlight 12 or excitation light 13 to pass through light-interruption plate122. Rotating shaft 121 causes light-interruption plate 122 to rotate toa particular position where the other of excitation light 12 orexcitation light 13 passes through open region 123.

Light-interruption plate 125, illustrated in FIG. 7B, has a semicircularshape. Light-interruption plate 125 is fixed to shaft 121, with which itrotates. Rotating shaft 121 causes light-interruption plate 125 torotate about the shaft, interrupting one of excitation light 12 orexcitation light 13 at a given time.

Optical chopper 120 may also include a light-interruption plate having adifferent shape than those described above. If sensing apparatus 601were, for example, to comprise more than two light sources, an opticalchopper having a shape other than a semicircle would be desirable.Moreover, it is possible to use plural optical choppers in a sensingapparatus.

Of course, it is possible, and may be preferable in some instances, toallow excitation light 12 and excitation light 13 to reach a singlelocation on sample 90 at the same time. Eliminating chopper 120 fromapparatus 601 would bring allow both excitation light 12 and excitationlight 13 to irradiate a location on sample 90 simultaneously. Generally,however, it is preferable to include chopper 120, or other knowncomponents, to allow only one of excitation light 12, 13 to reach alocation on sample 90 at a time.

Operation of sensing apparatus 601 will now be described, with referenceto FIGS. 2 and 8.

At a first point in time, optical source 10 outputs excitation light 12,which reflects off of optical filter 20, is received by optical assembly30, and is directed to a first location 91 on sample 90, as illustratedin FIGS. 8A and 4A. Chopper 120 interrupts excitation light 13,preventing it from reaching sample 90.

Sample 90 emits light 50 having a first wavelength in response toexcitation light 12. Emitted light 50 passes through optical filters 20,22, and 60 and focusing lens 70 before reaching detector 80.

At a second point in time, as illustrated in FIG. 8B, chopper 120 movesto a second position, at which optical chopper allows excitation light13 to pass, but interrupts excitation light 12. At this second point intime, optical assembly 30 has rotated about axis 45 to a secondposition, as illustrated in FIGS. 8B and 4B. In this instance,excitation light 13 is output by optical source 11, reflects off ofoptical filter 22, passes through optical filter 20 and optical assembly30, and reaches a second location 92 on sample 90.

Sample 90 emits light 61 having a second wavelength in response toexcitation light 13. Emitted light 61 passes through optical filters 20,22, and 60 and focusing lens assembly 70 before reaching detector 80.

In sensing apparatus 601, optical filter 20 is configured to allow lighthaving the wavelengths of excitation light 13, emitted light 50, andemitted light 61 to pass through it. Optical filter 20, however, isconfigured to reflect light having the wavelength of excitation light12. Optical filter 22 is configured to reflect light having thewavelength of excitation light 13, but is configured to allow lighthaving the wavelengths of emitted light 50, 61 to pass. Thus, afterpassing through optical filters 20 and 22, emitted light 50, 61 shouldcontain substantially only light emitted by sample 90. To further reducethe amount of excitation light 12 and excitation light 13 that may reachdetector 80, optical filter 60 is optionally provided to furtherattenuate wavelengths other than those emitted by sample 90.

Detector 80, as used in sensing apparatus 601, detects both wavelengthsof light emitted by sample 90, and typically cannot distinguish emittedlight 50 from emitted light 61. Optionally, however, sensing apparatus601 may employ additional equipment to discriminate between the twooptical signals. For example, a computer controlling detector 80 may usethe rate at which chopper 120 rotates to determine whether detector 80received emitted light 50 or emitted light 61 at a particular time. Bycorrelating the light received by detector 80 with the excitation light12, 13 incident on sample 90 at that time, the computer can determinewhat materials were present on sample 90.

As illustrated, for example, in FIG. 2, preferably sample 90 moves in alinear direction 95 in relation to axis 45 of optical assembly 30. InFIG. 2, sample 90 and sample holder 100 move in direction 95 while theremainder of sensing apparatus 601 remains substantially stationary,other than the rotation of optical assembly 30 and optical chopper 120.Sample 90, however, may remain stationary while rotating opticalassembly 30 moves in a linear direction relative to sample 90. In eithercase, the speed of the linear relative movement of sample 90 and axis 45preferably remains constant and slow compared to the rotational speed ofoptical assembly 30.

As described above and illustrated, for example, in FIG. 10, therotation of optical assembly 30 about axis 45 defines rotational sensingcircle 200. The radius of rotational sensing circle 200 extends fromaxis 45 to axis 46. Preferably, rotational sensing circle 200 has adiameter larger than a width of sample 90 perpendicular to direction ofthe linear relative motion represented by arrow 95 in FIG. 10.

Over time, the relative motion of sample 90 and axis 45 causes eachlocation on sample 90 to be illuminated by a leading arc 201 of therotational sensing circle 200, a trailing arc 202 of the rotationalsensing circle 200, or by both leading arc 201 and trailing arc 202.“Leading” arcs and “trailing” typically arcs occupy substantiallyopposite sides of sensing circle 200, and are located such that thedirection of linear relative motion of sample 90, shown by arrow 95 inFIG. 10, points from the “trailing” arc toward the “leading” arc. Forexample, arrow 95 points from trailing arc 202 toward leading arc 201 inFIG. 10. By illuminating multiple regions on sample 90 and capturingpixels corresponding to light detected from each region on sample 90,sensing apparatus 601 can create an image of the light emitted by sample90 using one of the methods described with regard to sensing apparatus600 above.

The linear relative movement of sample 90 with respect to rotationalsensing circle 200 will cause sensing apparatus 601 to illuminate sample90 with a sequence of leading arcs 201 i, 201 ii, 201 iii, and a seriesof trailing arcs 202 i, 202 ii, and 202 iii. The distance betweenconsecutive arcs, such as leading arcs 201 i, 201 ii, and 201 iii,depends on the number of revolutions of the focal point of objectivelens assembly 40 per unit length of the linear relative movement indirection 95. The displacement between consecutive arcs, such as leadingarcs 201 i, 201 ii, and 201 iii, defines the resolution of sensingapparatus 601 in direction 95.

Varying the combination of excitation light 12, 13 present when leadingarc 201 and trailing arc 202 pass over sample 90, provides severaloperational modes for sensing apparatus 601. These different operationalmodes, examples of which are illustrated in FIGS. 11-15 and describedbelow, provide different ways to provide excitation light 12, 13 to alllocations on sample 90. In each mode of operation, sample 90 iscompletely covered by illuminated arcs, or parts of arcs, stemmingeither from the leading arc 201, trailing arc 202, or both incombination. Allowing only one of excitation light 12, 13 to reachsample 90 during the complete scanning operation constitutessingle-channel scanning. Conversely, allowing both excitation light 12and excitation light 13 to reach sample 90, whether simultaneously, insequence, or another combination, during the complete scanning operationconstitutes dual-channel scanning. Persons of skill in the art willappreciate that adding more excitation sources will similarly providefor increasing levels of multi-channel scanning.

In each of the exemplary operational modes described below, sensingapparatus 601 directs illumination light 12 and/or illumination light 13to sample 90 and detects emitted light 50 and/or emitted light 61 fromsample 90. To simplify the discussion herein, the description of FIGS.11-15 below describes only the illumination of sample 90. It is to beunderstood, however, that these exemplary operational modes also allowfor the detection of light emitted by sample 90 using detector 80.

FIG. 11 illustrates a “normal” single-channel mode, in which excitationlight 12 illuminates sample 90 only during leading arc 201. When sensingapparatus 601 operates in the normal single channel mode, excitationlight 13 is not supplied to sample 90 during any portion of the fullrotational sensing circle 200. Exposing all locations on sample 90 toexcitation light 12 requires relative linear relational movement ofsample 90 and axis 45 over a distance d1. This relative linear movementcauses sample 90 to move from a first position, which FIG. 11 indicateswith solid lines, to a second position, shown with dashed lines 210.

An alternate normal single-channel mode is provided by illuminatingsample 90 with excitation light 13 during the leading arc 201, but notexposing sample 90 to excitation light 12 during the entire rotationalsensing circle 200. Moreover, replacing the leading arc 201 with thecorresponding trailing arc 202 will require a length of linearrelational movement equal to distance d1 to completely illuminate sample90. Sensing Apparatus 600, which comprises a single optical source, canalso operate in the normal single-channel mode shown in FIG. 11.

When the number of revolutions per unit of linear relational movementremains constant, distance d1 is directly proportional to the timerequired to expose all locations on sample 90 to excitation light 12.The same relationship between time and total relative linear movementexists for the operational modes illustrated in FIGS. 12, 13, 14, and15, if the number of revolutions per unit of linear relational movementremains constant. Comparing the distance traveled by sample 90 in eachof these figures allows a comparison of the time necessary to illuminateall locations on sample 90 with excitation light using differentoperational modes for sensing apparatus 601. Namely, requiring sample 90to travel a longer distance, corresponds to more time required to exposeall of sample 90 to excitation light.

FIG. 12 illustrates a “fast” single-channel mode, where excitation light12 reaches sample 90 during both the leading arc 201 and trailing arc202. As in the normal single channel mode illustrated in FIG. 11,excitation light 13 is not sent to sample 90 at any time during theoperation of sensing apparatus 601. Sending excitation light 12 to alllocations on sample 90 requires linear relational movement of sample 90and axis 45 over a distance d2, moving sample 90 to the positionindicated by dashed lines 211 in FIG. 12.

As mentioned above, when the number of revolutions per unit of linearrelational movement remains constant, the distance traveled by sample 90is directly proportional to the time required to expose all locations onsample 90 to illumination light 12, 13. Since sample 90 is exposed toexcitation light 12 over both leading arc 201 and trailing arc 202 (FIG.12), more of sample 90 is exposed during each rotation of opticalassembly 30 than if exposed over only leading arc 201 (FIG. 11). Thus,distance d1, the distance traveled by sample 90 in FIG. 11, is greaterthan distance d2, the distance traveled by sample 90 in FIG. 12. SensingApparatus 600, which comprises a single optical source, can also operatein the fast single-channel mode shown in FIG. 12.

Together, FIGS. 13A and 13B illustrate a normal dual-channel mode,showing, in FIG. 13A, the complete illumination of sample 90 in one ofthe two normal single-channel modes described above, followed by acomplete scanning of sample 90 in the other of the two normalsingle-channel modes described above in FIG. 13B. In this manner,sensing apparatus 601 senses sample 90 using both optical source 10 (inFIG. 13A) and optical source 11 (in FIG. 13B). The length of thecomplete dual channel scan is distance d3, being twice the distance d1.

During the first sensing operation, sample 90 moves from its originalposition to a second position, shown by dashed lines 212 in FIG. 13A.The sample subsequently moves to a third position, represented by dashedlines 213 in FIG. 13B. Sample 90 is not, however, limited to themovement illustrated in FIGS. 13A and 13B. The movement required by thenormal dual-channel mode may be accomplished using two separate sensingapparatuses 601 (or sensing apparatuses 600). Alternatively, sample 90may be moved in a first linear direction during the first sensingoperation and moved in the opposite the direction during the secondsensing operation. Moreover, the normal dual-channel mode can beaccomplished by performing the first sensing operation on sample 90,then moving sample 90 back to its original position before beginning thesecond sensing operation. No matter which of these movements of sample90 is used, sample 90 will move an entire distance d3 while beingsensed.

The normal dual-channel mode of operation has several disadvantagesrelative to other modes of operating sensing apparatus 601. Inparticular, the normal dual-channel mode of operation is the slowest ofthe dual-channel modes. Moreover, the sequential illumination oflocations on sample 90 with excitation light 12, 13 are delayed relativeto one another, providing the possibility of excitation source ordetector drift. The normal dual-channel mode, however, has theadvantages of avoiding crosstalk between channels and using a singledetector 80 instead of separate detectors for light emitted in responseto each of excitation light 12, 13. (A sensing apparatus 602 havingmultiple detectors 80, 81 will be discussed below.)

When operating in the fast dual-channel mode, illustrated in FIG. 14,sensing apparatus 601 first illuminates sample 90 using one of the twofast single-channel modes described above (for example with opticalsource 10) and then illuminates sample 90 in the other of the two fastsingle-channel modes described above (for example with optical source11). Sample 90 moves from its initial position to the position indicatedby dashed lines 214 in FIG. 14A, and then to the position indicated bydashed lines in FIG. 14B. As with the normal dual-channel mode discussedabove, sample 90 is not limited to the particular motion illustrated inFIGS. 14A and 14B. The movement of sample 90 may be accomplished byreversing its direction, returning sample 90 to its original positionbefore beginning the second sensing operation, using multiple sensingapparatuses, or other methods.

In this fast dual-channel mode, sample 90 moves a total distance d4,which is twice the distance d2. Distance d4 is less than distance d3,and thus sample 90 can be sensed faster by using two fast single-channelmodes in sequence than using two successive normal single-channel modes.

In the fast dual-channel mode of operation, a delay exists betweensequential illuminations of locations on sample 90 with excitation light12, 13 relative to one another, creating the possibility of excitationsource or detector drift. Because only excitation light 12 or excitationlight 13 illuminates sample 90 during each sensing of sample 90, thefast dual-channel mode eliminates potential crosstalk between channelsand requires only a single detector 80 instead of dual matcheddetectors. Moreover, the fast dual-channel mode of operation completesits sensing operation more quickly than the normal dual-channel modediscussed above.

FIG. 15 depicts an overlaid dual-channel mode. When sensing apparatus601 operates in this overlaid dual-channel mode, excitation light 12exposes sample 90 during leading arc 201 and excitation light 13 exposessample 90 during trailing arc 202. To completely illuminate alllocations on sample 90 in this mode of operation, sensing apparatus 601requires a linear relational movement over distance d5, moving sample 90to the position indicated by dashed lines 216 in FIG. 15. Moving sample90 in this manner provides two separate complete illuminations of sample90, each covering sample 90 completely by arcs, or parts of arcs.Because excitation light 12 exposes sample 90 during leading arc 201 andexcitation light 13 exposes sample 90 during trailing arc 202, alllocations on sample 90 receive both excitation light 12 and excitationlight 13.

Distance d5 is less than either distance d3 or distance d4. Thus, usingthe overlaid dual-channel mode provides faster illumination of alllocations on sample 90 than using either the normal dual-channel mode orthe fast dual-channel modes. Another advantage of the overlaiddual-channel mode is the absence of crosstalk between channels duringscanning, as leading arc 201, which illuminates sample 90 withexcitation light 12, and trailing arc 202, which illuminates sample 90with excitation light 13, are physically displaced from each other. Apossible disadvantage of the overlaid dual-channel mode is thepreexposure of sample 90 by excitation light 12 during the leading arc201 in relation to the following displaced scanning by the excitationlight 13 during the trailing arc 202.

An interleaved dual-channel mode will now be described with reference toFIG. 12. In this interleaved dual-channel mode, excitation light 12reaches sample 90 during both the leading arc 201 and the trailing arc202 during a first revolution of the objective lens assembly 40.Excitation light 13 does not expose sample 90 during this firstrevolution of objective lens assembly 40. In a second revolution ofobjective lens assembly 40, however, the on/off status of opticalsources 10 and 11 is switched. Thus, during, the second revolution ofobjective lens assembly 40, excitation light 13 illuminates sample 90,but excitation light 12 does not. Preferably, the first and secondrevolutions represent consecutive revolutions of objective lens assembly40, and the pattern is repeated so that sensing apparatus 601 createstwo complete sensings of sample 90 based on alternating revolutions,each of the two sensings being illuminated by a different one of the twoexcitation lights 12, 13.

Using this operational mode, sample 90 moves a distance d2, from itsoriginal position to the position indicated by dashed lines 211.

Because the arcs are interleaved in the manner described above, thisoperational mode results in half of the resolution of the other dualscanning operational modes. To compare the interleaved dual-channel modeto the other dual scanning modes, therefore, it is necessary to doubledistance d2. Doubling distance d2 results in a distance equal todistance d4, shown in FIG. 14.

The interleaved dual-channel operational mode provides a sensing speedequal to that of the fast dual-channel mode. The dual channeloperational mode does not, however, create a significant time spanbetween the sensing of each channel. Thus, little drift of theexcitation sources and detector occurs between channel scans when usingthe interleaved dual-channel operational mode.

Finally, FIG. 12 can also be used to describe a simultaneousdual-channel operational mode. In this simultaneous dual channeloperational mode, sensing apparatus 601 illuminates sample 90 with bothexcitation light 12 and excitation light 13 turned on during both theleading arc 201 and the trailing arc 202. As detector 80 will notseparate emitted light 50 from emitted light 61, the output from sensingapparatus 601 will represent the sum of fluorescence resulting from bothexcitation light 12 and excitation light 13.

When operating in this simultaneous dual-channel operational mode,sensing apparatus 601 only requires sample 90 to move over distance d2.

Distance d2 is less than distance d3, distance d4, and distance d5,indicating that using the simultaneous dual-channel operational mode isfaster than either of the other dual-channel modes. It is noted,however, that not all applications are suited to work with only a singlecombined output from the two excitation sources. FIG. 9 illustratessensing apparatus 603, consistent with further aspects of the invention.Sensing apparatus 603 comprises the same components as sensing apparatus601, except for blocking filters 130, 131 are provided instead ofchopper 120.

Blocking filter 130 preferably is configured to block light having thewavelength of excitation light 12, while blocking filter 131 preferablyblocks light having the wavelength of excitation light 13. Blockingfilters 130, 131, when combined with the rotation of optical assembly 30about axis 45, allow only one of excitation light 12, 13 to reach sample90 at a time.

As illustrated in FIG. 9A, for example, at a first point in time whenoptical assembly is in a first position, blocking filter 130 preventsexcitation light 11 from reaching sample 90. At a second point in timeafter optical assembly 130 rotates to a second position, blocking filter131 prevents excitation light 13 from reaching sample 90, as illustratedin FIG. 9B. In this manner, blocking filters 130, 131 allow only one ofexcitation light 12, 13 to reach sample 90 at any given time.

Blocking filters 130, 131 constrain the operational modes available tosensing apparatus 603, as blocking filters 130, 131 effectively limitleading arc 201 to one of excitation light 12, 13 and trailing arc 202to the other of excitation light 12, 13. Thus, blocking filters 130, 131prevent the use of the fast single-channel mode, the normal dual-channelmode, the fast dual-channel mode, and the interleaved dual-channel mode.Sensing apparatus 603 can, however, operate in the normal single-channelmode and the overlaid dual-channel operation mode.

FIG. 3 illustrates sensing apparatus 602, consistent with furtheraspects of the present invention. As illustrated in FIG. 3, sensingapparatus 602 comprises the same components as sensing apparatus 601.Sensing apparatus 602, further comprises, however, additional opticalfilters 24, 64, an additional focusing lens assembly 71, and anadditional detector 81.

In sensing apparatus 602, optical filter 24 separates emitted light 50and emitted light 61, by reflecting emitted light 61, for example, whilepassing emitted light 50. As a result, emitted light 50 is directedtoward detector 80 and emitted light 61 is directed toward detector 81.

Optical filter 64, focusing lens 72, and pin hole 73 are configured toreduce noise and focus emitted light 61 before it reaches opticaldetector 81. Optical filter 64, focusing lens 72, and pin hole 73operate in a manner similar to optical filter 60, focusing lens 70, andpin hole 71, respectively. Optical filter 64, focusing lens 72, and pinhole 73 are optionally provided to enhance performance of sensingapparatus 602.

Emitted light 61 is converted to corresponding electrical signals bydetector 81, which a user or computer can store and/or analyze. Likedetector 80, detector 81 may comprise, for example, a photo multiplyingtube and an image sensor such as a charge-coupled device (CCD).

Like sensing apparatuses 600 and 601, sensing apparatus 603 can createan image representing the light emitted by sample 90 by illuminatingmultiple regions on sample 90 and capturing pixels corresponding tolight detected from each region. Moreover, sensing apparatus 602 canoperate in any of the operational modes described above with respect tosensing apparatus 601. Because sensing apparatus 602 contains twoseparate detectors 80, 81, however, sensing apparatus 602 can operate inan additional operational mode not discussed above, the separatesimultaneous dual-channel mode, to be described with reference to FIG.12. The separate simultaneous dual-channel mode allows illumination ofsample 90 with both excitation sources turned on during both the leadingarc 201 and the trailing arc 202. Due to the presence of multipledetectors 80, 81, sensing apparatus 602 can distinguish emitted light 50from emitted light 61. Sensing apparatus 602 can thus output twosimultaneous, complete sensings of sample 90, one from each detector 80,81.

In this operational mode, sample 90 only moves a distance d2 relative toaxis 45. The separate simultaneous dual-channel mode is as fast orfaster than any of the other dual-channel modes described above.Moreover, the separate simultaneous dual-channel mode has the additionaladvantage of using separate detectors 80, 81 for each channel, making itable to functionally replace any of the other dual-channel modes ofoperation.

The presence of separate detectors 80, 81, however, makes sensingapparatus 602 prone to crosstalk between channels. Moreover, sensingapparatus 602 requires more components than sensing apparatus 601,including additional detector 81, additional optical filters 24, 64, andadditional focusing lens assembly 71. The presence of these additionalcomponents increases manufacturing and maintenance costs. FIG. 16illustrates a control circuit 300, which may be used instead of chopper120 in any embodiment of the present invention requiring a chopper.Control circuit 300 provides allows apparatuses 601, 602 to selectivelyactivate optical sources 10, 11 in response to the rotation of opticalassembly 30 through leading arc 201 and trailing arc 202.

Control Circuit 300 utilizes electronic control input 314 to selectivelyactivate optical source 10 and electronic control input 315 toselectively activate optical source 11. Electronic control inputs 314,315 allow control circuit 300 to independently set the on or off stateof optical sources 10, 11 by respectively setting the control inputs314, 315 to on or off.

A plate 303 rotates in synchronization with optical assembly 30, indirection 301. Preferably, plate 303 is mounted on optical rotatingassembly 30, with axis 45 passing through a central hole 302 on plate303. Excitation light 12, 13 passes through hole 302 before enteringoptical assembly 30. As shown in FIG. 16, optical assembly 30 preferablyis fixed at an angle after the beginning of opening 305, the anglecoverage of opening 305 being larger than the sensing arc across thewidth of sample 90.

Plate 303 has an opening 305, which allows an optical fork sensor 308 tosense when optical assembly 30 passes through leading arc 201. Opticalfork sensor 308 remains stationary while plate 303 and optical assembly30 rotate. Optical fork sensor 308 outputs a sense signal of “one”during leading arc 201, when it senses light passing from its dedicatedlight source through cutout 305.

Digitizer disk 303 further also has a second opening 306, which allows asecond optical fork sensor 307 to sense when optical assembly 30 passesthrough trailing arc 202. Optical fork sensor 307 remains stationary asplate 303 and optical assembly 30 rotate, sensing light passing from itsdedicated light source through cutout 306. Optical fork sensor 307outputs a sense signal of “one” during trailing arc 202.

As mentioned above, control circuit 300 is configured such that opticalfork sensor 308 outputs a “one” sense signal during leading arc 201.Thus, for example, if switch circuit 316 is closed during leading arc201, the sense signal from optical fork sensor 308 will travel tocontrol input 314, causing optical source 10 to output excitation light12.

The status of switch circuits 316, 317, 318 and 319 is controlled by 4bits of the addressed output location of memory 323. A “one” sets thecorresponding switch circuit on, and a “zero” turns it off. Thus, theaddressed output location of memory 323 indicates whether the activationof optical sources 10, 11 is desired. A sequencer 320 controls thelocation in memory 323 that is read out to the switch circuits, throughaddress bus 322. Sequencer 320 receives signals from optical fork sensor308 via control input 321. When optical fork sensor 308 transitions froma sense signal of “zero” to a sense signal of “one,” sequencer 320increments the memory addresses in memory 323, changing the 4 outputbits from memory 323 to the next 4 bits in a prestored sequence. In thismanner, the memory address is incremented once per revolution of opticalassembly 30, at the start of leading arc 201.

Switch circuits 316, 317, 318 and 319 control whether optical sources10, 11 receive signals from optical fork sensors 307, 308. Thus, controlcircuit 300 controls which of excitation light 12, 13 illuminates sample90 during leading arc 201 and trailing arc 202. Accordingly, controlcircuit 300 turns optical sources 10, 11 on and off to selectivelysupply excitation light 12, 13 to sample 90.

Memory 323, sequencer 320, and address bus 322 have at least thecapacity to store and output in sequence one 4-bit word for each of therevolution of the plate 33 required to conduct and complete the sensingof sample 90. A sequence of 4-bit output words from memory 323 can, forexample, include all words having identical content, which would keepone or both optical sources 10, 11 on or off during the completescanning operation. Also, it is possible that each 4-bit word in thesequence differs from the preceding 4-bit word, thereby allowing one orboth of optical source 10 and optical source 11 to be switched on or offat each revolution during the complete scanning operation.

Optical sources 10, 11 are thus independently controlled by the contentof memory 323 via the electronic control inputs 314 and 315 during bothleading arc 201 and trailing arc 202. Electronic chopper 300 may beconfigured to allow excitation light 12, 13 to reach sample 90 duringleading arc 201, trailing arc 202, both arcs, or neither arc.Combinations of excitation light 12, 13 during the leading arc 201 andtrailing arc 202 provide for the various operational modes describedabove.

FIG. 5 illustrates a flow chart of a sensing method 700, consistent witha further aspect of the present invention.

In Step 710, excitation light is generated, preferably, where thewavelength is selected to cause a particular material in a sample tofluoresce, as noted above.

In Step 715, the optical assembly is rotated to a first position.Preferably, step 715 includes rotating the optical assembly about anaxis. Moreover, it is preferable that the optical assembly isconstructed in manner consistent with optical assembly 30, describedabove.

Next, in step 720, the excitation light is directed through the opticalassembly to a first location on the sample. Preferably, the excitationlight enters the optical assembly along the axis about which the opticalassembly is rotated in step 715 and leaves the optical assembly along asecond, substantially parallel axis. The excitation light may, however,enter the optical assembly along a different axis.

It is desirable for the sample to include a biochip or other samplecontaining multiple testing sites.

Preferably, between step 720 and 725, sensing method 700 furtherincludes receiving first emitted light—emitted by the sample—with theoptical assembly, collimating the first emitted light, and directing thefirst emitted light toward a detecting device before detecting the firstemitted light. Moreover, consistent with the apparatuses describedabove, sensing method 700 may include filtering and focusing the firstemitted light just before step 725.

In step 725, the first emitted light is detected. The first location onthe sample fluoresces and emits this first emitted light in response tothe excitation light. Upon detecting the first emitted light, the methodmay include converting the first emitted light into an electric signaland storing or further processing that electric signal.

Next, in step 730, the optical assembly is rotated to a second position.Step 730 preferably includes rotating the optical assembly about theaxis mentioned in conjunction with step 715 above.

In step 735, the excitation light is directed through the opticalassembly to a second location on the sample. This step differs from step720 in that the optical assembly now is in the second position, insteadof the first position, causing the excitation light to be directed tothe second location on the sample, instead of the first location on thesample.

Between steps 735 and 740, sensing method 700 may further includereceiving second emitted light—emitted by the sample from the secondlocation—with the optical assembly, collimating the second emittedlight, and directing the second emitted light toward a detecting devicebefore detecting the second emitted light. Sensing method 700 may alsoinclude filtering and focusing the second emitted light before step 740.

Finally, in step 740 the second emitted light is detected. As in step725, step 740 may include converting the first emitted light into anelectric signal and storing or further processing that electric signal.

Method 700 may further comprise moving the sample. Preferably, themoving step includes moving the sample in a linear directionsubstantially perpendicular to the axis about which the optical assemblyis rotated. The moving step is preferably performed in a step-wisemanner. A continuous, non-linear moving step may also be incorporatedinto method 700.

Method 700 may further include the utilization of one of the operationalmodes described above. For example, the first location specified in step715 may be located on a first arc and the second location specified instep 730 may be located on a second arc. The first arc and second arcmay be located on substantially opposite sides of a circle defined bythe rotation of the optical assembly, the first and second arcs eachspanning a width of the sample substantially perpendicular to a lineardirection in which the sample moves. Selectively allowing excitationlight to illuminate the sample in one or both steps 720 and 735 allowsone to practice the single-channel modes of operation described above.

For example, the normal single-channel mode described in FIG. 11, wouldrequire that excitation light reach the sample only in step 720. Steps730, 735, and 740 would be eliminated, and the method would includerotating the optical assembly to a second location on the arc anddirecting the excitation light to the second location on that arc.

As another example, to practice the fast single-channel mode, whereexcitation light 12 reaches sample 90 during both the leading arc 201and trailing arc 202, one would only need to modify method 700 toinclude the first and second arc described above.

Other adjustments necessary to practice each of the single-channel modeswill be apparent from the description of FIGS. 11-14 above.

FIG. 6 illustrates sensing method 800, another sensing method consistentwith the present invention.

In step 810, first and second excitation light are generated.Preferably, this step includes using a first laser to generate firstexcitation light and a second laser to generate second excitation light.The wavelengths of the first and second excitation light preferablydiffer, each being selected to cause a particular material to fluoresce.

In step 815, an optical assembly is rotated to a first position.Preferably, step 815 includes rotating the optical assembly about anaxis. The optical assembly preferably is constructed in a mannerconsistent with optical assembly 30, described above.

In step 820, the first and second excitation light is directed throughthe optical assembly to a first location on a sample. This step issubstantially similar to step 720 of method 700, except that step 820includes directing two excitation lights, instead of a single excitationlight. As in method 700, it is desirable for the sample to include abiochip or other sample containing multiple testing sites. Step 820 mayfurther comprise directing one of the first excitation light and thesecond excitation light to the sample at a time.

Preferably, between step 820 and 825, sensing method 800 furtherincludes receiving first and second emitted light—emitted by thesample—with the optical assembly, collimating the first emitted light,and directing the first emitted light toward a detecting device beforedetecting the first emitted light. Moreover, sensing method 800 mayinclude filtering and focusing the first emitted light just before step825.

In step 825, first and second emitted light are detected. The firstlocation on the sample emits first emitted light in response to thefirst excitation light and emits second emitted light in response to thesecond excitation light. Method 800 may further comprise converting thefirst and second emitted light into an electric signal or signals andstoring or further processing that electric signal or signals.Preferably, and consistent with sensing apparatus 602, described above,the first emitted light is detected with a different detector than thesecond emitted light.

Next, in step 830, the optical assembly is rotated to a second position.Step 830 preferably includes rotating the optical assembly about theaxis mentioned in conjunction with step 815 above.

In step 835, the first and second excitation lights are directed throughthe optical assembly to a second location on the sample. This stepdiffers from step 820 in that the optical assembly now is in the secondposition, instead of the first position, causing the first and secondexcitation light to be directed to the second location on the sample,instead of the first location on the sample. Step 835 may furthercomprise directing one of the first excitation light and the secondexcitation light to the sample at a time.

Between steps 835 and 840, sensing method 800 may further includereceiving third and fourth emitted light—emitted by the sample from thesecond location—with the optical assembly, collimating the third andfourth emitted light, and directing the third and fourth emitted lighttoward a detecting device before detection. Moreover, sensing method 800may include filtering and focusing the third and fourth emitted lightjust before step 840.

Finally, step 840 comprises detecting the third and fourth emittedlight. As in step 825, step 840 may include converting the first emittedlight into an electric signal and storing or further processing thatelectric signal.

Method 800 may further comprise moving the sample. Preferably, themoving step includes moving the sample in a linear directionsubstantially parallel to the axis about which the optical assembly isrotated. The moving step is preferably performed in a step-wise manner.A continuous, non-linear moving step may also be incorporated intomethod 800.

Method 800 may further include the utilization of one of the operationalmodes described above. For example, the first location specified in step815 and the second location specified in step 830 may be located on afirst arc and a second arc, respectively. The first arc and second arcmay be located on substantially opposite sides of a circle defined bythe rotation of the optical assembly, and spana width of the samplesubstantially perpendicular to a linear direction in which the samplemoves. Selectively allowing first and second excitation light toilluminate the sample in each of steps 820 and 835 allows one topractice the dual-channel modes of operation described above.

For example, to practice the simultaneous dual-channel operational mode,where first and second excitation light are directed to the sample onboth leading arc 201 and trailing arc 202 (See FIG. 12), method 800 needonly be amended to include the arcs discussed above.

To practice the overlaid dual-channel mode illustrated in FIG. 15, forexample, one would amend method 800 to direct only first excitationlight to the sample in step 820 and direct only second excitation lightto the sample in step 835. Moreover, step 825 would be amended toinclude detecting only first emitted light and step 840 would be amendedto include only detecting second emitted light, emitted in response tosecond excitation light.

Other adjustments necessary to practice each of the dual-channel modeswill be apparent from the descriptions of FIGS. 11-15 above.

The apparatuses and sensing methods described above allow users toquickly direct and receive light from distinct locations on samples. Theapparatuses and sensing methods detect light emitted by samplescontaining multiple test sites arranged in a non-rectilinear patternsand can detect emitted light without moving the samples in rectilinearpattern. Moreover, samples used with the apparatuses and sensing methodsdescribed can comprise non-rectangular test sites.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. As anexample, excitation light 12 (and excitation light 13) may enter opticalassembly 30 along an axis different than axis 45. Moreover, for example,additional optical sources, each producing laser beams having uniquewavelengths, could be added to the sensing apparatus, enabling itmeasure more than two experimental results.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

1. A sensing method comprising: generating excitation light; rotating anoptical assembly in one direction to a first position; directing theexcitation light into the optical assembly along the optical assembly'srotational axis directing the excitation light out of the opticalassembly along a second axis, wherein the optical assembly's rotationalaxis and the second axis are substantially parallel; directing theexcitation light through the optical assembly to a first location on asample; detecting first emitted light; rotating the optical assembly inthe one direction to a second position; directing the excitation lightthrough the optical assembly to a second location on the sample;detecting second emitted light; and again rotating the optical assemblyin the one direction to the first position.
 2. The sensing method ofclaim 1, wherein the sample includes a biochip.
 3. The sensing method ofclaim 1, further comprising moving the sample.
 4. A sensing methodcomprising: generating first and second excitation light; rotating anoptical assembly in one direction to a first position; directing thefirst and second excitation light into the optical assembly along theoptical assembly's rotational axis directing the first and secondexcitation light out of the optical assembly along a second axis,wherein the optical assembly's rotational axis and the second axis aresubstantially parallel; directing the first and second excitation lightthrough the optical assembly to a first location on a sample; detectingfirst and second emitted light; rotating the optical assembly in the onedirection to a second position; directing the first and secondexcitation light through the optical assembly to a second location onthe sample; detecting third and fourth emitted light; and again rotatingthe optical assembly in the one direction to the first position.
 5. Thesensing method of claim 4, further comprising directing one of the firstexcitation light and the second excitation light to the sample at atime.
 6. The sensing method of claim 3, wherein the moving includesmoving the sample in a linear direction.